A modified X10-23 DNAzyme that can better access large, structured RNA targets.

The 10-23 DNA enzyme is one of the most efficient RNA-cleaving enzymes reported, possessing substrate recognition arms that can be designed to target virtually any AU diribonucleotide junction. However, 10-23 often shows reduced activity for large, structured RNA (lsRNA) substrates like messenger RNA. Increasing arm length or adding antisense DNA oligonucleotides (ASOs) can improve accessibility to lsRNA but may also reduce the efficiency of product release. Xeno nucleic acids (XNAs), such as 2'-fluoro-arabinonucleic acid (FANA), have been substituted for DNA into the arms of 10-23 to improve activity, such as in the FANA-modified X10-23, but X10-23 also shows poor accessibility for lsRNA targets. To overcome this issue, we substituted patterns of various XNAs with high RNA binding strength into the substrate recognition arms of X10-23. We found that an X10-23 enzyme with a distinct 2'F-RNA-LNA-FANA arm pattern, denoted as XdZ-2, could gain access to several lsRNA targets from SARS-CoV-2, achieving cleavage rates up to 82-fold faster than X10-23 for one system. While the ASO strategy provided higher cleavage rates for two other lsRNA systems, XdZ-2 may be a more attractive alternative in low Mg2+ environments and in terms of improving the efficiency of product release and stability in biological samples.